This experiment was conducted to investigate a gradient centrifugation separation method for microorganisms in gastrointestinal contents and faeces of swine. First, dilute the gastrointestinal contents and faeces with separation buffer to find the fittest dilution ratio. Then gradiently centrifuge and acquire microbial bacteria from gastrointestinal contents and faeces samples. By this method, the best dilution ratio of gastro, jejunum and ileum contents to separation buffer ranged from 8.0% to 12.0%, and the weight of samples ranged from 3.2 to 4.8 g. The best dilution ratio of cecum, colon contents and faeces ranged from 2.0% to 8.0%, and the weight ranged from 0.8 to 1.6 g. We detected the amino acids composition of microorganisms in ileum and faeces as well as the diversity of microorganisms in gastrointestinal content and faeces of weighed at 30 kg pigs fed with three kinds of protein level diets (12.0%, 15.0% and 18.0%). The results showed that the most amino acids composition of microbes in ileum contents and faeces were significantly responsible to different protein levels of nutritional model (P<0.05). The bands in polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) figure were rich and legible with microorganisms separated from gastrointestinal contents and faeces. The study demonstrates that this method has high separation degree and obtains substantial dry cell weight. Meanwhile it owns advantages of less cost, easy operation and good repeatability, which provides a method for studying the physiological function of gastrointestinal microbes deeply.